Proteomics

Dataset Information

0

MS-based sequencing of the anti-FLAG-M2 antibody

ABSTRACT: We demonstrate a method for direct de novo sequencing of monoclonal IgG from the purified antibody products. The method uses a panel of multiple complementary proteases to generate suitable peptides for de novo sequencing by LC-MS/MS in a bottom-up fashion. Furthermore, we apply a dual fragmentation scheme, using both stepped high-energy collision dissociation (stepped HCD) and electron transfer high-energy collision dissociation (EThcD) on all peptide precursors. The method achieves full sequence coverage of the monoclonal antibody Herceptin, with an accuracy of 98% in the variable regions. We applied the method to sequence the widely used anti-FLAG-M2 mouse monoclonal antibody, which we successfully validated by remodeling a high-resolution crystal structure of the Fab and demonstrating binding to a FLAG-tagged target protein in Western blot analysis. The method thus offers robust and reliable sequences of monoclonal antibodies.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human) Mus Musculus (mouse)

SUBMITTER: Joost Snijder  

LAB HEAD: Joost Snijder

PROVIDER: PXD023419 | Pride | 2021-01-11

REPOSITORIES: Pride

Json Xml
altmetric image

Publications

Mass Spectrometry-Based <i>De Novo</i> Sequencing of Monoclonal Antibodies Using Multiple Proteases and a Dual Fragmentation Scheme.

Peng Weiwei W   Pronker Matti F MF   Snijder Joost J  

Journal of proteome research 20210614 7

Antibody sequence information is crucial to understanding the structural basis for antigen binding and enables the use of antibodies as therapeutics and research tools. Here, we demonstrate a method for direct <i>de novo</i> sequencing of monoclonal IgG from the purified antibody products. The method uses a panel of multiple complementary proteases to generate suitable peptides for <i>de novo</i> sequencing by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in a bottom-up fashion. Furt  ...[more]

Similar Datasets

Proteomics De Novo Sequencing of SARS-CoV-2 and influenza monoclonal antibodies by mass spectrometry using HCD and EThcD fragmentation and Supernovo software
2022-06-03 | PXD030094 | Pride
Proteomics Sequencing the anti-MUC1 hybridoma antibody 139H2
2024-03-18 | PXD043489 | Pride
Proteomics Enrichment and Fragmentation Approaches for Enhanced Detection and Characterization of Endogenous Glycosylated Neuropeptides
2022-08-19 | PXD033226 | Pride
Proteomics EThcD-sceHCD-MS/MS-based Clinical N-glycoproteomics
2022-04-04 | PXD030288 | Pride
Proteomics Direct mass spectrometry-based detection and antibody sequencing of Monoclonal Gammopathy of Undetermined Significance from patient serum
2023-09-14 | PXD042266 | Pride
Proteomics Electron-based dissociation is needed for O-glycopeptides derived from OpeRATOR proteolysis
2020-11-03 | PXD020077 | Pride
Proteomics Intact Transition Epitope Mapping- Thermodynamic Weak-force Order (ITEM-TWO)
2019-11-07 | PXD016024 | Pride
Proteomics Stradard protein and yeast top-down data
2022-06-09 | PXD022941 | Pride
Proteomics Mass Spectrometric Analysis of Antibody-Epitope Peptide Complex Dissociation: Theoretical Concept and Practical Procedure of Binding Strength Characterization
2020-10-29 | PXD021296 | Pride
Proteomics Optimal Dissociation Methods Differ for N- and O-glycopeptides
2020-07-20 | PXD017646 | Pride